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www.sciencemag.org/content/354/6307/102/suppl/DC1
Supplementary Material for
An artificial metalloenzyme with the kinetics of native enzymes
P. Dydio, H. M. Key, A. Nazarenko, J. Y.-E. Rha, V. Seyedkazemi, D. S. Clark, J. F. Hartwig*
*Corresponding author. Email: [email protected]
Published 7 October 2016, Science 354, 102 (2016)
DOI: 10.1126/science.aah4227
This PDF file includes:
Materials and Methods Figs. S1 to S12 Tables S1 to S7 References
2
Materials and Methods
I. Protein Expression, Purification, and Characterization
a. General Methods
Unless otherwise noted, the chemicals, salts, and solvents used were reagent grade
and used as received from commercial suppliers without further purification. All
expression media and buffers were prepared using ddH2O (MilliQ A10 Advantage
purification system, Millipore). All expression media were sterilized using either an
autoclave (45 min, 121oC) or a sterile syringe filter (0.22 um). To maintain sterile
conditions, sterile materials and E. coli cells were manipulated near a lit Bunsen burner.
b. Genes and Cloning
The WT CYP119 gene cloned into the vector 2BT (6xHis-TEV-ORF; AddGene
#29666) was purchased from GenScript with codon optimization for E. coli (Table S1).
The genes for WT P450 BM3 and P450-CAM were obtained as a gift from Prof. Humin
Zhao (University of Illinois) and cloned to the vector 2BT at the QB3 Macrolab at UC
Berkeley.
c. Media Preparation
Preparation of optimized minimal expression media: Salts (15 g Na2HPO4, 7.5 g
K2HPO4, 0.3 g NaH2PO4, 0.3 g KH2PO4, 1.5 g NaCl, 5.0 g NH4Cl) were dissolved in 2 L
ddH2O and autoclaved to give a media with pH ~8.0 - 8.2. Solutions of glucose (20%),
casamino acids (BD Company, low Fe, 20%), and MgSO4 (1 M), were autoclaved
separately. Solutions of ampicillin (100 mg/ mL) and CaCl2 (1 M) were sterilized by
syringe filter. The following amounts of the listed solutions were added per 2 L of sterile
salt solution: 40 mL glucose, 20 mL casamino acids, 4 mL MgSO4, 100 uL CaCl2, 2 mL
ampicillin. Stock solutions were stored for several weeks; prepared media was stored for
less than 1 day. Minimal media plates were prepared from the same media with the addition
of 17 g agar/L media. In this case, agar was autoclaved in 1 L ddH2O, and salts were
autoclaved separately as a 20X solution, after which they were added to the agar solution.
d. Mutagenesis
Site-directed mutagenesis was performed using the QuickChange Lightening
mutagenesis kit (Agilent); requisite double stranded DNA primers were designed
according to the Agilent Primer Design Program and purchased from Integrated DNA
Technology. PCR reactions were performed according to the manufacturer’s directions.
PCR reactions contained 5 uL reaction buffer, 34 uL ddH2O, 1.5 uL QuickSolution, 1 uL
plamids (50 ng/uL), 1.25 uL sense primer (100 ng/uL), 1.25 uL antisense primer (100 ng/
uL), 5 uL dNTPs (2 mM/base), and 1 uL polymerase.
PCR Program: Phase 1 (1 cycle): 95 °C, 1.5 min; Phase 2 (18 cycles): 95 °C, 20 sec,
60 °C, 10 sec, 68 °C, 4.5 min; Phase 3 (1 cycle): 68 °C, 3 min; Phase 4 (storage): 4 °C.
DNA Isolation and Storage: Following the completion of the above set of PCR
procedures, 1.5 uL DPN 1 was added to each reaction, and the reactions were further
incubated (3 h, 37 °C). The crude PCR mixture was used to transform XL-10 Gold
3
Ultracompetent cells (45 L cells, 2 L PCR reactions). The mixture was incubated on ice
(30 min), heat shocked (30 s, 42 °C), recovered with SOC media (1 h, 37 °C, 275 rpm),
and plated on LB plates. Plates were grown (18 h, 37 °C), and individual colonies were
used to inoculate 1 mL rich media cultures, which were grown in 96-well plates (13 h, 37
°C, 300 rpm). DNA was isolated from the 96-well cultures using magnetic bead technology
at the UC Berkeley DNA Sequencing Facility. Alternatively, individual colonies were used
to inoculate 4 mL rich media cultures and grown overnight (13 h, 37 °C, 300 rpm), and the
plasmids were purified using a Qiagen DNA Miniprep kit according to the manufacturer’s
instructions.
e. Protein Expression
Optimized Expression of Apo CYP119: BL21 Star competent E. coli cells (50 L,
QB3 Macrolab, UC Berkeley) were thawed on ice, transferred to 14 mL Falcon tubes, and
transformed with the desired plasmid solution (2 L, 50-250 ng/L). The cells were
incubated on ice (30 min), heat shocked (20 sec, 42°C), re-cooled on ice (2 min), and
recovered with SOC media (37 °C, 1 h, 250 rpm). Aliquots of the cultures (50uL) were
plated on minimal media plates (expression media supplemented with 17 g agar/L) and
incubated (20 h, 37 °C) to produce approximately 10-100 colonies per plate. Single
colonies were used to inoculate starter cultures (3 mL, expression media), which were
grown (4-8 h, 37°C, 275 rpm) and used to inoculate 100 mL overnight cultures (minimal
media, 37° C, 275 rpm). Each culture grown overnight was used to inoculate 750 mL of
minimal media, which was then grown further (9 h, 37 °C, 275 rpm). Expression was
induced with IPTG (800 uL, 1M), and the cultures were grown further (15 h, 30 °C, 275
rpm). Cells were harvested by centrifugation (5000 rpm, 15 min, 4° C), and the pellets were
resuspended in 20 mL Ni-NTA lysis buffer (50 mM NaPi, 250 mM NaCl, 10 mM
Imidazole, pH = 8.0) and stored at 80 °C until purification.
Protein Purification: Cell suspensions were thawed in a room-temperature water bath,
decanted to 50 mL glass beakers, and lysed on ice by sonication (3x30 sec on, 2x2 min off,
65% power). Cell debris was removed by centrifugation (10 000 rpm, 30 min, 4 °C), and
Ni-NTA (5 mL, 50% suspension per 850 mL cell culture) was added. The lysates were
briefly incubated with Ni-NTA (30 min, rt, 20 rpm) and poured into glass frits (coarse, 50
mL). The resin was washed with Ni-NTA lysis buffer (3 x 35 mL), and the wash fractions
were monitored using a Bradford assay dye. The desired protein was eluted with 18 mL
Ni-NTA elution buffer (50 mM NaPi, 250 mM NaCl, 250 mM Imidazole, pH = 8.0),
dialyzed twice against Tris buffer (10 mM, pH = 8.0, 1 h, rt), concentrated to the desired
concentration using a spin concentrator, and metallated within several hours. Apo protein
was not stored for more than 8 hours.
Metallation of the Apo-Protein (General Method): Stock solutions of metal cofactors
in DMF were added to solutions of apo protein (0.12 mM) at room temperature in the
desired stoichiometry with a final DMF concentration of 2%. The proteins were briefly
incubated at room temperature (5 minutes), and DMF was removed by using a NAP column
equilibrated with the reaction buffer (100 mM NaPi, 100 mM NaCl, pH = 6.0).
4
Protein Storage: Glycerol was added to a solution of the protein (3:1 v:v protein
solution: 50% glycerol), the solution was divided into 1.5 mL Eppendorf tubes (0.5 mL per
aliquot), and the tubes were flash frozen in liquid nitrogen and stored at 80 °C until further
use. Frozen aliquots of the protein were thawed in a room-temperature water bath.
f. Protein Characterization
Gel Electrophoresis: Protein purity was analyzed by sodium dodecyl sulfate-
polyacrylamide (SDS-PAGE) gel electrophoresis using precast gels (polyacrylamide, 10-
20% linear gradient, Biorad).
Mass Spectrometry: Apo-proteins were analyzed with an Agilent 1200 series liquid
chromatograph connected in-line with an Agilent 6224 time-of-flight (TOF) LC/MS
system using a Turbospray ion source. Metallated proteins were analyzed by native
nanoelectrospray ionization mass spectrometry (nanoESI-MS) using a Waters Q-ToF
Premier quadrupole time-of-flight mass spectrometer equipped with a nanoESI source
(Milford, MA). Mass spectra were acquired in the positive ion mode and processed using
MassLynx software (version 4.1, Waters). The instrument is located in the QB3/Chemistry
Mass Spectrometry Facility at UC Berkeley.
Melting Temperature: The melting temperature of Ir(Me)PIX containing heme
proteins were determined by differential scanning fluorimetry (DSF) using SYPRO Orange
as the fluorescent reporter, according to an established protocol. DSF curves were obtained
using a CFX96 Touch Real Time PCR Detection System (BioRad), and data were
processed using the Solver extension of Microsoft Excel. Conditions and melting
temperatures can be found in Table S7. DSF curves can be found in Figure S1.
II. Organic Synthesis and Characterization
a.General methods and materials
Unless stated otherwise, all reactions and manipulations were conducted on the
laboratory bench in air with reagent grade solvents. Reactions under inert gas atmosphere
were carried out in the oven dried glassware in a nitrogen-filled glovebox or by standard
Schlenk techniques under nitrogen.
NMR spectra were acquired on 400 MHz, 500 MHz, 600 MHz, or 900 MHz Bruker
instruments at the University of California, Berkeley. NMR spectra were processed with
MestReNova 9.0 (Mestrelab Research SL). Chemical shifts are reported in ppm and
referenced to residual solvent peaks(26). Coupling constants are reported in hertz. Chiral
SFC analysis was conducted on a JASCO SF-2000 integrated analytical SFC system. GC
analyses were obtained on an Agilent 6890 GC equipped with either, an HP-5 column (25
m x 0.20 mm ID x 0.33 m film) for achiral analysis or Cyclosil-B column (30m x 0.25mm
x 0.25 m film) for chiral analysis, and an FID detector. GC yields were calculated using
dodecane as the internal. High-resolution mass spectra and elemental analysis were
obtained via the Micro-Mass/Analytical Facility operated by the College of Chemistry,
University of California, Berkeley.
5
Unless noted otherwise, all reagents and solvents were purchased from commercial
suppliers and used without further purification. If required, dichloromethane (DCM) and
tetrahydrofuran (THF) were degassed by purging with argon for 15 minutes and dried with
a solvent purification system containing a one-meter column of activated alumina; dried
and degassed acetonitrile, 1,2-xylene, toluene, N,N-dimethylformamide (DMF), ethanol
and methanol were purchased form commercial suppliers and used as received.
b. Substrates
The synthetic procedures and characterization of ethyl (2-methoxyphenyl)-
diazoacetate (1), methyl (2,5-dimethoxyphenyl)diazoacetate (S3), methyl (2,3-
dimethoxyphenyl)diazoacetate (S4), (2-ethoxyphenyl)-diazoacetate (S6) (12) and methyl
(2-benzyloxyphenyl)diazoacetate (S7) (27) were reported previously.
Methyl (5-chloro-2-methoxyphenyl)diazoacetate (S5):
In a closed vial, a solution of 5-chloro-2-methoxyphenylacetic acid (4.0 g, 20 mmol)
in methanol (15 ml) containing several drops of sulfuric acid, was stirred overnight at 80
°C. The volatile materials were evaporated under vacuum. The residue was dissolved in
ethyl acetate (~40 ml), washed with NaHCO3 sat. (40 ml) and water (40 ml), dried over
MgSO4 and evaporated. The crude product was used in the next step without further
purification.
To a stirred solution of methyl 5-chloro-2-methoxyphenylacetate (20 mmol) and 4-
acetamidobenzenesulfonyl azide (p-ABSA, 7.2 g, 30 mmol) in acetonitrile (40 ml) at 0 °C,
1,8-diazabicycloundec-7-ene (DBU, 4.8 ml, 32 mmol) was added dropwise. The cooling
bath was removed, and the reaction was allowed to continue stirring overnight. The
reaction mixture was diluted with dichloromethane (~60 ml), washed with water (2 x ~50
ml), dried over MgSO4 and evaporated. The crude product was purified by column
chromatography on silica gel, with a mixture of hexanes and ethyl acetate (100:0 – 90:10
gradient) as the eluent. Fractions of the pure product were combined, and the solvent
evaporated, yielding 3.9 g (81%) of product. 1H NMR (500 MHz, CDCl3): d = 7.55 (d, J = 2.7 Hz, 1H), 7.15 (dd, J = 8.8, 2.6 Hz, 1H),
6.76 (d, J = 8.8 Hz, 1H), 3.80 (s, 3H), 3.80 (s, 3H); 13C NMR (151 MHz, CDCl3): d = 166.19, 153.85, 129.29, 127.97, 126.32, 115.57,
112.03, 112.02, 55.99, 52.20 (C=N2 signal missing, as observed before for related
molecules(28);
6
HR MS (EI): calcd. for C10H9N2O4Cl [M]+: 240.0302, found: 240.0303.
Methyl (2-ethylphenyl)diazoacetate (7):
Methyl (2-ethylphenyl)acetate was prepared following a synthetic procedure
developed for analogous compounds(23): to a stirred solution of 2-bromobenzyl acetate (2
g, 8.7 mmol) in 30 mL of THF were added freshly prepared ethylzinc bromide (20 mL,
~0.6-0.7 M in THF)(29) and Pd(t-Bu3P)2 (90 mg, 0.175 mmol) sequentially at room
temperature. The reaction mixture was stirred at room temperature for 8 h, then quenched
with 1N HCl (30 mL). The product was extracted with ethyl acetate (3 x 50 mL), and the
combined organic layers were washed with brine (30 mL), dried over MgSO4 and
evaporated. The crude product was purified by column chromatography on silica gel, with
e eluent. Fractions of
the pure product were combined, and the solvent evaporated, yielding 1.29 g (83%) of
product as colorless liquid. 1H NMR (500 MHz, CDCl3): 7.21 – 7.08 (m, 4H), 3.65 (s, 3H), 3.63 (s, 2H), 2.62 (q, J =
7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H); 13C NMR (151 MHz, CDCl3): d = 172.36, 142.76, 132.15, 130.63, 128.74, 127.78,
126.22, 52.20, 38.62, 25.98, 15.02;
HR MS (EI): calcd. for C11H14O2 [M]+: 178.0994, found: 178.0997.
To a stirred solution of methyl (2-ethylphenyl)acetate (1.29 g, 7.1 mmol) and 4-
acetamidobenzenesulfonyl azide (p-ABSA, 2.4 g, 10 mmol) in acetonitrile (40 ml) at 0 °C,
1,8-diazabicycloundec-7-ene (DBU, 1.8 ml, 12 mmol) was added dropwise. The cooling
bath was removed, and the reaction was allowed to continue stirring for 48 h (the reaction
progress was followed by TLC). The reaction mixture was diluted with dichloromethane
(~50 ml), washed with water (2 x ~50 ml), and dried over MgSO4. After filtration, the
volatile material from the filtrate was evaporated under reduced pressure. The crude
product was purified by column chromatography on silica gel, with a mixture of hexanes
and ethyl acetate (100:0 – 95:5 gradient) as the eluent. Fractions of the pure product were
combined, and the solvent evaporated, yielding 0.72 g (50%) of product. 1H NMR (400 MHz, CDCl3): d = 7.37 – 7.17 (m, 4H), 3.79 (s, 3H), 2.61 (q, J = 7.6 Hz,
2H), 1.21 (t, J = 7.5 Hz, 3H); 13C NMR (151 MHz, CDCl3): d = 166.89, 144.17, 131.71, 129.58, 128.96, 126.47,
123.74, 52.25, 26.33, 14.55 (C=N2 signal missing, as observed before for related
molecules(28)
HR MS (EI): calcd. for C11H12N2O2 [M]+: 204.0899, found: 204.0899.
c. Authentic Products
General procedure for synthesis of dihydrobenzofurans
To 5 ml of a solution of a derivative of methyl (2-methoxyphenyl)diazoacetate (~50
mM) in toluene 60-300 ul of a solution of Ir(Me)-PIX (8 mM, 0.2-1 mol%) in DMF was
added, and the reaction mixture was stirred vigorously. The reaction progress was
7
monitored by TLC. Upon completion, the volatile materials were evaporated under reduced
pressure, and the residue was purified by column chromatography on silica gel, with a
mixture of hexanes and ethyl acetate (100:0 – 80:20 gradient) as eluent. Fractions of the
pure product were combined, and the solvent evaporated, yielding 20-90% of desired
product. The NMR data match those of the reported molecules: methyl 2,3-
dihydrobenzofuran-3-carboxylate (2)(29), methyl 5-methoxy-2,3-dihydrobenzofuran-3-
carboxylate (3)(12), 7-methoxy-2,3-dihydrobenzofuran-3-carboxylate (4), (12) 5-chloro-
2,3-dihydrobenzofuran-3-carboxylate (5)(21), methyl trans-2-methyl-2,3-
dihydrobenzofuran-3-carboxylate (trans-6)(12), methyl trans-2-phenyl-2,3-
dihydrobenzofuran-3-carboxylate (trans-7)(27), and methyl cis-2-phenyl-2,3-
dihydrobenzofuran-3-carboxylate (cis-7)(27).
Methyl indane-1-carboxylate (11):
In a closed vial, a solution of methyl (2-ethylphenyl)diazoacetate (50 mg) and Ir(Me)-
PIX (~1 mg) in toluene (10 ml) was stirred at 80 °C. The reaction progress was monitored
by TLC. Upon completion (~16 h), the volatile materials were evaporated under reduced
pressure, and the residue was purified by column chromatography on silica gel, with a
mixture of hexanes and ethyl acetate (100:0 – 95:5 gradient) as eluent. Fractions of the
pure product were combined, and the solvent evaporated, yielding title product. The NMR
data match those of the reported molecule(30).
d. Ir(Me)-Mesoporphyrin IX (Ir(Me)-PIX) cofactor used in the studies:
The synthetic procedures and characterization of Ir(Me)-PIX were reported
previously(12).
III. Catalytic Experiments
a. General Methods:
Unless otherwise noted, catalytic reactions were performed in 4 mL individually-
capped vials or in 1.2 mL vials as part of a 96-well array fitted with a cover that is attached
by screws. Reactions were either (1) assembled in a nitrogen atmosphere glove box or (2)
assembled on the bench. In the latter case, the headspace of the vial was purged with
8
nitrogen through a septum cap. Solutions of Ir(Me)-PIX-CYP119 were gently degassed on
a Schlenk line (3 cycles vacuum/refill) before being pumped into a glove box in sealed
vials. Organic reagents were added as stock solutions in DMF, such that the final amount
of DMF in the reaction was approximately 2% by volume (unless noted otherwise). Protein
catalysts were diluted to reaction concentrations in sodium phosphate buffer (100 mM, pH
= 6.0) before being added to reaction vials. Unless otherwise noted, all reactions were
performed with catalysts generated from a 1:2 ratio of Ir(Me)-cofactor : apo protein, with
0.17 mol % catalyst loading, based on the ratio limiting reagent to metal cofactor. Unless
otherwise noted, all reactions were conducted in a shaking incubator (20 °C, 16 h, 275
rpm).
b. Procedure for typical catalytic experiments
The catalyst stock solution: The Ir(Me)-CYP119 catalyst was prepared by addition of
a stock solution of Ir(Me)-PIX (3.1 mM in DMF) to a solution of the apo-protein (0.13mM
in 10 mM Tris buffer, pH = 8), such that the resulting solution had a 1 : 2 ratio of Ir(Me)-
PIX : CYP119. This ratio was used to ensure that all Ir-PIX was bound. The mixture was
incubated for 5 min, and desalted with the NAP-10 desalting column equilibrated with
reaction buffer (100 mM NaPi, 100 mM NaCl, pH = 6.0). The protein mixture was diluted
to the required reaction concentration with the same reaction buffer.
Intramolecular C-H Insertion: 250 ul of the catalyst stock solution (0.0425 umol of
[Ir]) was added to a vial, followed by addition of a stock solution of the appropriate diazo
compound (2.5 umol in 5 uL DMF). For experiments with higher substrate concentration
(> 50 mM), the substrate was added directly to a vial, followed by the appropriate organic
solvent and 250 ul of the catalyst stock solution. The vial was sealed with a cap, removed
from a glovebox and incubated in a shaker (20 °C, 275 rpm). For experiments at elevated
temperatures, the vial was incubated in a metal heat block at the reported temperature.
Upon completion, the reaction mixture was analyzed as described in section IV.
Intermolecular C-H Insertion: 1 mL of the catalyst stock solution (0.017 mM Ir(Me)-
CYP119 in 100 mM NaPi, 100 mM NaCl, pH =6.0) was added to a 4 mL vial containing
a micro stir bar. The vial was capped with a cap containing a septa, and the catalyst solution
was degassed by three cycles of vacuum/nitrogen refill, after which it was tranfered to a
nitrogen atmosphere wet box. In the box, 10 umol phthalan (1.1 uL) and 10 uL DMF were
added to the vial, and a separate solution of EDA was prepared in DMF (50X relative to
final targeted EDA concentration). The reaction vial and EDA solution were covered with
septa caps and removed from the glovebox. A syringe pump outfitted with a series of gas
tight syringes was used to add the solution of EDA (20 uL) to each reaction vial over 1
hour. After the conclusion of the reaction, the reaction mixture was quenched and analyzed
as described in section IV.
c. Mutant Screening (directed evolution)
Mutants were evaluated for C-H insertion reactions in 96-well plates with the reaction
conditions described in the general method (Section Va,b). Twelve mutants were analyzed
per each 96-well plate. Aliquots of each Ir(Me)-P450 mutant were distributed down the
columns of a 96-well plate. Next, aliquots of substrates for intramolecular C-H insertion
9
were added to each of the rows (A-E) to generate up to 60 unique reactions. The reaction
with the most selective mutants were repeated. The same enantioselectivities and similar
yields were measured in both sets of experiments. The results are reported in Tables S5.
d. Kinetic experiments
The initial rates of selected mutants for C-H insertion reactions were evaluated in 96-well
plates with the catalyst stock solution prepared as described in the general method section
(Section Va,b). The stock solutions of the substrate in different concentrations (2 – 100
mM in DMF) were distributed in the first row of a 96-well plate. Aliquots of 250 ul of each
solution of Ir(Me)-P450 mutant (0.001 or 0.005 mM, depending on the activity of the
variant) or Ir(Me)-myo mutant (0.005 mM) or free cofactor Ir(Me)-PIX (0.001 mM), were
distributed to remaining rows of a 96-well plate(rows B-H). Next, 12.5 ul aliquots of the
substrate stock solutions from row A were added to each of the rows containing catalyst
solutions (B-H) to initiate reactions. After suitable period of time (2-80 min, depending on
the activity of the variant), the reactions were simultaneously quenched by the addition
HBr (60 uL, 50% in water). All manipulations were performed using a multichannel pipet.
The reaction mixtures were analyzed as described in section IV. (Similar
enantioselectivities were measured as in the experiments conducted under standard
conditions). The measured yields were used to calculate reaction rates. Repeated
experiments, including experiments with different time points and different catalyst
concentrations resulted in similar reaction parameters for the same variants of Ir(Me)-
CYP119. The kinetic parameters of the enzymes were obtained by the data fitting with the
standard Michaelis-Menten kinetic model, using the data fitting software package Origin
8.0. The results are reported in Figures 2 (main text) and Figure S11 and Table S2.
e. Reactions on a synthetic scale
The catalyst stock solution – prepared as in described section Vb – was added to a
Schlenk flask and gently degassed on a Schlenk line (3 cycles vacuum/refill). A solution
of the appropriate diazo substrate in DMF was added (the concentration of the stock
solution adjusted such that the final amount of DMF in the reaction was approximately 2%
by volume). The flask was sealed and gently agitated by shaking (120 rpm) or end over
end rotation (20 rpm) at 20 °C. The reaction was diluted with brine (30 ml) and extracted
with ethyl acetate (3·50 ml). If required, the phase separation was achieved by centrifuging
(2000 rpm, 3 min) the mixture. The combined organic fractions were washed with brine
(30 ml) and dried over MgSO4. After filtration, the volatile material from the filtrate was
evaporated under reduced pressure. The residue was purified by column chromatography
on silica gel, with a mixture of hexanes and ethyl acetate (100:0 – 90:10 gradient) as the
eluent. Fractions of the pure product were combined, and the solvent evaporated. The
specific conditions (variants of the catalyst, loadings, and substrate concentrations), yields,
enantioselectivites and specific rotations of products for reactions with different substrates
are reported in Table S3.
IV. Analysis of Yield and Enantiomeric Ratio (er)
Yields were determined by achiral or chiral GC using dodecane as an internal standard
(Figure S2). Enantiomeric ratios were determined either by chiral GC or chiral SFC
10
(Figures S2-S10). Achiral GC was used to determine the yields for reactions analyzed by
SFC, while the yields for reactions analyzed by chiral GC were determined concurrently
with that analysis. The methods used to determine the e.r. of each product are summarized
in Table S6. Representative traces can be found in Figures S2-S10. Samples for analysis
were prepared as follows, depending on the analysis method:
SFC/achiral GC: Saturated NaCl (200 uL) was added to each reaction vial, followed
by a solution of dodecane (500 ul, 1 ul/ml) in EtOAc. The contents of the vial were mixed
by pipet, and the phases were allowed to separate. A portion (250 uL) of the organic layer
was removed from the top of the vial by pipet, transferred to a new vial, evaporated and
redissoved in MeOH for analysis by SFC. An additional portion of EtOAc (250 uL) was
added to the original reaction vial, and the reaction was quenched by the addition of HBr
(60 uL, 50% in water). After separation of the layers, approximately 400 uL of the aqueous
phase was removed from the bottom of the vial by pipet. The remaining contents of the
vial were neutralized by the addition of sat. NaHCO3 (200 uL), and the organic layer was
further diluted with EtOAc (500 uL). The organic layer was then transferred to a separate
vial for GC analysis. In the case of experiments performed in a 96-well array, all
manipulations were performed using a multichannel pipet.
Chiral GC: Saturated NaCl (200 uL) was added to each reaction vial, followed by a
solution of dodecane (500 ul, 1 ul/ml) in EtOAc. The contents of the vial were mixed by
pipet, and the phases were allowed to separate. The reaction was then quenched by the
addition HBr (40 uL, 50% in water). After separation of the layers, approximately 400 uL
of the aqueous phase was removed from the bottom of the vial by pipet. The remaining
contents of the vial were neutralized by the addition of sat. NaHCO3 (200 uL), and the
organic layer was further diluted with EtOAc (500 uL). The organic layer was then
transferred to a separate vial for GC analysis. In the case of experiments performed in a
96-well array, all manipulations were performed using a multichannel pipet.
11
V. Supplementary Figures
Deconvoluted mass spectrum of apo CYP119-C317G acquired by LC-MS:
Native nanoESI-MS of Ir(Me)-PIX CYP119, showing (+14), (+13), and (+12) charge
states. In each charge state, the initial (small) shoulder corresponds to the apo-protein,
the main signal corresponds to the mass of the Ir(Me)-protein, and the third (small) signal
corresponds to the adduct of a second Ir(Me)-PIX to the protein.
Differential scanning fluorimetry curves used to determine the melting temperature of
Ir(Me)-PIX containing heme proteins:
Fig. S1.
Characterization of CYP119 protein by liquid chromatography/mass spectrometry (mass
determination, above) and differential scanning fluorimetry (melting temperature, below)
12
y = 0.5179xR² = 0.9975
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.5 1 1.5 2
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound 2
y = 0.7264xR² = 0.9874
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.2 0.4 0.6 0.8 1 1.2 1.4
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound 3
y = 0.7216xR² = 0.9724
0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound 4
13
y = 0.6425xR² = 0.9932
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 0.5 1 1.5 2
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound 5
y = 0.7238xR² = 0.9998
0
0.2
0.4
0.6
0.8
1
1.2
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound 6
y = 1.0702xR² = 0.9969
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 0.2 0.4 0.6 0.8 1 1.2 1.4
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound cis-7
14
y = 1.1133xR² = 0.9996
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound trans-7
y = 0.8738xR² = 0.9999
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.2 0.4 0.6 0.8 1 1.2
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound 9
y = 0.7644xR² = 0.9944
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.2 0.4 0.6 0.8 1 1.2
rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound 11
15
Fig. S2
Calibration curves for products 3-7, 9 and 11-13.
y = 0.3154xR² = 0.9999
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.5 1 1.5 2rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound trans-12
y = 0.3112xR² = 0.9999
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 0.5 1 1.5 2rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound cis-12
y = 0.1189xR² = 0.996
0
0.05
0.1
0.15
0.2
0.25
0.3
0 0.5 1 1.5 2 2.5rati
o o
f p
eak
ares
fo
r an
alyt
e to
d
od
ecan
e
molar ratio of analyte to dodecane
compound 13
16
Fig. S3
Chiral GC traces for product 2; racemate (above) and enantioenriched sample (below).
17
Fig. S4
Chiral GC traces for product 3; racemate (above) and enantioenriched sample (below).
18
Fig. S5
Chiral GC traces for product 4; racemate (above) and enantioenriched sample (below).
19
Fig. S6
Chiral GC traces for product 5; racemate (above) and enantioenriched sample (below).
20
Fig. S7
Chiral GC traces for product 6; racemate (above) and enantioenriched sample (below).
21
Fig. S8
Chiral SFC traces for product 7; racemate (above) and enantioenriched sample of cis-
7(middle), and trans-7 (below).
22
Fig. S9
Chiral GC traces for product 9; racemate (above) and enantioenriched sample (below).
23
Fig. S10
Chiral SFC traces for product 11; racemate (above) and enantioenriched sample (below).
24
y = 6.1987xR² = 0.9856
0
0.5
1
1.5
2
2.5
3
3.5
0 0.1 0.2 0.3 0.4 0.5 0.6
rate
/ m
in-1
[substrate]
free cofactor Ir(Me)-PIX
free cofactor
Linear (free cofactor)
25
Ir(Me)-CYP119 WT – no satisfactory fit to the Michaelis-Menten kinetic model was
obtained, presumably because the Km value is too high for this enzyme:
26
Ir(Me)-CYP119- C317G:
27
Ir(Me)-CYP119- C317G,T213A:
28
Ir(Me)-CYP119-Max (C317G,T213G,L69V,V254L) – data from one time point:
29
Ir(Me)-CYP119-Max (C317G,T213G,L69V,V254L) - data from multiple
experiments (multiple time point and catalyst concentrations):
30
Ir(Me)-myoglobin-H93A, H64V:
Fig. S11
Plots of initial rates for reactions of substrate 1 to form 2 in the presence of different
variants of Ir(Me)-CYP119, selective variant of Ir(Me)-mOCR-myoglobin (H93A,
H64V) and free cofactor Ir(Me)-PIX. Comparison of the activity of different enzymes
(above) and data and the fit to the Michaelis-Menten model for each reaction(below).
31
Fig. S12
Enantioselectivities and TOFs of C-H insertion reactions catalyzed by Ir(Me)-PIX
CYP119-MAX supported on separose activated with cyanogen bromide (CNBr). Catalyst
preparation: 0.017 umol of Ir(Me)-CYP119-Max was supported on 0.1 mL of CNBr resin,
according to the manufacturer’s instructions. The resin was suspended in 0.25 mL NaPi,
pH = 6.0 containing 100 mM NaCl, and 2.5 umol substrate was added as a solution in 5 uL
of DMF. After 1 hour, the resin was collected by gravity filtration, washed with 10 column
volumes of reaction buffer and re-subjected to a subsequent reaction under identical
conditions. The solution (containing the product) collected upon filtering the catalyst from
the reaction was analyzed as described in section IV.
32
VI. Supporting Tables
Protein Organism Constructi
on Vector Sequence
mOCR-Myo-globin
Physeter macrocephalus
6xHis-TEV-mOCR-Myo
2BT EGDIHMKSSHHHHHHENLYFQSNMSNMTYNNVFDH
AYEMLKENIRYDDIRDTDDLHDAIHMAADNAVPHY
YADIRSVMASEGIDLEFEDSGLMPDTKDDIRILQA
RIYEQLTIDLWEDAEDLLNEYLEEVEEYEEDEEGT
GSETPGTSESGVLSEGEWQLVLHVWAKVEADVAGH
GQDILIRLFKSHPETLEKFDRFKHLKTEAEMKASE
DLKKHGVTVLTALGAILKKKGHHEAELKPLAQSHA
TKHKIPIKYLEFISEAIIHVLHSRHPGDFGADAQG
AMNKALELFRKDIAAKYKELGYQG
P450 BM3
Bacillus megaterium
P450-BM3-6xHis
pcWori MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIAD
ELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK
NLSQALKFARDFAGDGLVTSWTHEKNWKKAHNILL
PSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHI
EVSEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFI
ISMVRALDEVMNKLQRANPDDPAYDENKRQFQEDI
KVMNDLVDKIIADRKARGEQSDDLLTQMLNGKDPE
TGEPLDDGNIRYQIITFLIAGHEATSGLLSFALYF
LVKNPHVLQKVAEEAARVLVDPVPSYKQVKQLKYV
GMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK
GDEVMVLIPQLHRDKTVWGDDVEEFRPERFENPSA
IPQHAFKPFGNGQRASIGQQFALHEATLVLGMMLK
HFDFEDHTNYELDIKETLTLKPKGFVVKAKSKKIP
LGGIPSPSTHHHHHH
P450 CAM
Pseudomonas putida
P450-CAM-6xHis
pcWori MTTETIQSNANLAPLPPHVPEHLVFDFDMYNPSNL
SAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQ
LIREAYEDYRHFSSECPFIPREAGEAYDFIPTSMD
PPEQRQFRALANQVVGMPVVDKLENRIQELACSLI
ESLRPQGQCNFTEDYAEPFPIRIFMLLAGLPEEDI
PHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIE
QRRQKPGTDAISIVANGQVNGRPITSDEAKRMCGL
LLVGGLDTVVNFLSFSMEFLAKSPEHRQELIQRPE
RIPAACEELLRRFSLVADGRILTSDYEFHGVQLKK
GDQILL
PQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSH
LCLGQHLARRIIVTLKEWLTRIPDFSIAPGAQIQH
KSGIVSGVQALPLVWDPATTKAVHHHHHH
CYP 119 Sulfolobus solfataricus
6xHis-TEV-CYP119
2BT EGDIHMKSSHHHHHHENLYFQSNAMYDWFSEMRKK
DPVYYDGNIWQVFSYRYTKEVLNNFSKFSSDLTGY
HERLEDLRNGKIRFDIPTRYTMLTSDPPLHDELRS
MSADIFSPQKLQTLETFIRETTRSLLDSIDPREDD
IVKKLAVPLPIIVISKILGLPIEDKEKFKEWSDLV
AFRLGKPGEIFELGKKYLELIGYVKDHLNSGTEVV
SRVVNSNLSDIEKLGYIILLLIAGNETTTNLISNS
VIDFTRFNLWQRIREENLYLKAIEEALRYSPPVMR
TVRKTKERVKLGDQTIEEGEYVRVWIASANRDEEV
FHDGEKFIPDRNPNPHLSFGSGIHLCLGAPLARLE
ARIAIEEFSKRFRHIEILDTEKVPNEVLNGYKRLV
VRLKSNE
Table S1.
Sequence information for heme proteins used in this study.
33
# enzyme kcat (min-1) Km (mM) kcat/Km (min-1mM-1)
1 Ir(Me)-CYP119 WT - > 5 -
2 Ir(Me)-CYP119-C317G 0.22 ± 0.01 3.1 ± 0.3 0.071
3 Ir(Me)-CYP119-C317G,T213A 4.8 ± 0.4 0.40 ± 0.03 12
4 Ir(Me)-CYP119-Max
(C317G,T213G,L69V,V254L) - single experiment
45.8 ± 1.8 0.17 ± 0.03 269
5 Ir(Me)-CYP119-Max
(C317G,T213G,L69V,V254L) - all data
45.9 ± 1.7 0.20 ± 0.03 230
6 Ir(Me)-myoglobin H93A, H64V 0.73 ± 0.03 1.1 ± 0.2 0.66
Table S2.
Michaelis-Menten parameters for the carbene insertion of substrate 1 catalyzed by
variants of Ir(Me)-CYP119 and Ir(Me)-myoglobin.
34
Subs. Scale Catalyst (variant) Vol. Time Yield ee [L]D
20
1 20.6
mg
0.17 mol%
C317G, T213A, L155W
10 ml 24 h 5 mg of
(R)-2
+81% -22.0o
(c=0.5
CHCl3)
1 40 mg 0.05 mol% (317G,
L69V, T213G, V254L)
10 ml 24 h 18 mg of
(S)-2
-92%
1 206 mg 0.017 mol% (317G,
L69V, T213G, V254L)
50 ml 16 h 107 mg of
(S)-2
-92%
1 1000
mg
0.017 mol% (317G,
L69V, T213G, V254L)
250 ml 16 h 480 mg of
(S)-2 +
72 mg
rsmb
-93%
S3 35.4
mg
0.17 mol%
(317G, L69V, T213G,
A152W)
15 ml 24 h 28.6 mg
of 3
-94% +8.3o
(c=0.6
CHCl3)
S4 23.6
mg
0.17 mol%
(317G, T213G, L155F)
10 ml 24h 10.3 mg
of 4
+95%
S5 18.0
mg
0.17 mol%
(317G, L69V, T213G,
A152W)
7.5 ml 24 h 10.0 mg
of (S)-5a
(63%)
-85% -25.3o
(c=1.0
CHCl3)
S7 20.0
mg
0.17 mol%
(C317, V69W, T213G)
10 ml 24 h 10.0 mg
of cis-
(2S,3R)-
6a
+94% -56.8o
(c=0.5
CHCl3)
a Absolute configuration assigned based on the specific rotation – product 2(21) 5 and cis-7 (21, 27) b rsm = recovered starting material
Table S3.
Reactions of 2-7 on synthetic scale with variants of Ir(Me)-CYP119.
35
69X 209X 213X 254X
L 115-C10 116-G2 115-B12
V 115-D1 115-C2 116-F7
A 116-H3 115-B6
G 115-G4 115-C8 115-F2 115-E8
Y 115-D3 116-D1 115-B8
W 115-C12 116-F1 115-B10
F 115-D10 115-H8 115-B11
T 115-D2 117-A1
T213G + …
69X 209X 254X 310X 155X 152X
L 115-E3
V 116-B1
A 117-E4
G 115-G2 117-E7
Y 115-G5 116-E6 140-H7
W 116-A12 115-E2 140-B9
F 115-H1 115-E7 141-C1
T 115-G10 115-F7 115-E4 141-B1
69L 69V 69A 69G 69Y 69W 69F 69T
213L 116-B8 115-H9
213V 116-B10 116-A11 116-A10 116-B11
213A 116-B3 116-B6 115-H11 116-B5
213G
213Y 116-B7 116-A9 115-H4
213W 116-B4 116-A5
213F 115-H5 115-H2 115-H7
213T
254L 254V 254A 254G 254Y 254W 254F 254T
213L 116-D3 116-C10 116-C4
213V 116-C12
213A 116-C3 116-E8 116-C6
213G
213Y 116-D7
213W 116-E12 116-D12 116-E3
213F 116-E4 116-E10
213T
209L 209V 209A 209G 209Y 209W 209F 209T
213L 116-F2 116-G6
213V
213A
213G
213Y
213W 116-D2
213F
213T
209L 209V 209A 209G 209Y 209W 209F 209T
254L 117-C3 117-A10
254V
254A 117-A3
254G
254Y 117-A4 117-B3
254W 117-C5 117-B11
254F 117-C2 117-B7
254T
36
69L 69V 69A 69G 69Y 69W 69F 69T
209L 117-F6 117-G8 117-F5 117-G6 117-H12
209V 117-F9 117-H3 117-H7
209A
209G 117-H8 117-G4 117-G9
209Y
209W 117-H2 117-H6 117-F7 117-F11
209F
209T 117-G5 117-H4 117-H5 117-H10 117-G1
213G, 254L (115-E3) + Additional Mutation 69X 310X 318X
L 140-B10
V 140-B6
A 140-A3 140-D2
G 140-B3 140-B8
Y 140-A4 140-C5
W 140-B2
F 140-A8
T 140-C1
213G, 69Y (115-G5) +
69X 310X 318X
L 142-H4
V
A
G 140-C11
Y 142-H11
W 142-G7 140-D9
F
T 142-H12
T213A (116-H3) +
69X 254X 310X
L 166-A12
V
A
G 165-H1
Y 165-H3 166-A6
W
F 165-H6 166-A9
T 165-H7 166-A7
213G, 254L (115-E3) +
152X 155X
L 140-H1
V 141-A5 141-C10
A 141-B3
G 141-A9 141-C3
Y 140-H2 141-B11
W 141-A11 141-B2
F 140-H11 141-D3
T 140-G9 141-G4
213G, 69Y (115-G5) +
152X 155X
L
V 140-G8
A
G
Y 140-H10
W 140-H6
F
T
37
T213A (116-H3) +
318X 152X 155X
L 166-C7
V 166-B11 166-D2
A
G
Y 166-B1 166-C4 166-D7
W 166-B6 166-D5
F 166-B7 166-C3 166-D4
T 166-B3 166-D1
254X
69F, 213G 69V, 213G
L 141-F6 141-E9
V
A 141-E1
G
Y 141-D7 141-D9
W 141-F1 141-E3
F 141-E7
T
69W, 213G (116-A12) +
209X 254X 310X
L 165-A4 165-B6
V 165-A5 165-C11
A 165-B2 165-C10
G 165-A11 165-C1
Y 165-A7 165-B8
W 165-B3 165-C5
F 165-A2
T 165-B7 165-C2
69Y, 254L, 213G +
310X 155X
L
V
A
G
Y
W 140-B9 141-B12
F
T 141-B7
69W, 213G (116-A12) +
318X 152X 155X
L 165-E8 165-F11
V 165-D9
A
G 165-D3
Y 165-D7 165-E5 165-F5
W 165-D2 165-E7 165-F2
F 165-E1 165-F4
T 165-D1 165-F1
Table S4.
Plasmid library obtained from site directed mutagenesis of the CYP119 gene within the
2B plasmid in the course of directed evolution.
38
Table S5.
Complete, tabulated results for the directed evolution of Ir(Me)-CYP119 for
intramolecular C-H insertion reactions performed under the standard conditions for
screening the enzymes. The identity of the mutants can be found in Table S4. Blank cells
reflect that the mutant was not evaluated for the listed substrate.
39
Table S5.
Continued from previous page.
40
Table S5.
Continued from previous page.
41
Product Instrument Column Method Retention Times
2 GC CYCLOSIL-B
(30m x 0.25mm x
0.25u)
Isothermal: 135 °C t1(R)(-)=20.0 min
t2(S)(+)=20.5 min
3 GC CYCLOSIL-B
(30m x 0.25mm x
0.25u)
Isothermal: 145 °C t1(-)=44.7 min
t2(+)=45.6 min
3 SFC Chiracel OD-H
(Diacel)
Isocratic: 1%
MeOH, 4 ml/min
flow
t1(-)=2.3 min
t2(+)=3.4 min
4 GC CYCLOSIL-B
(30m x 0.25mm x
0.25u)
Isothermal: 165 °C t1(-)=19.5 min
t2(+)=20.0 min
5 GC CYCLOSIL-B
(30m x 0.25mm x
0.25u)
Isothermal: 150 °C t1(R)(-)=28.8 min
t2(S)(+)=29. min
6 GC CYCLOSIL-B
(30m x 0.25mm x
0.25u)
Isothermal: 150 °C t1(R)(-)=12.8 min
t2(S)(+)=12.9 min
7 SFC Chiracel OJ-H
(Diacel)
Isocratic: 1%
MeOH, 4 ml/min
flow
Trans:
t1=1.85 min
t2=2.1 min
Cis:
t1(2S,3R)(-)=4.6 min
t2(2R,3S)(+)=6.1 min
9 GC CYCLOSIL-B
(30m x 0.25mm x
0.25u)
Isothermal: 140 °C t1=13.1 min
t2=13.4 min
11 SFC Chiracel AZ-H
(Diacel)
Isocratic: 2%
MeOH, 4 ml/min
flow
t1=4.6 min
t2=5.3 min
Table S6.
Methods used to separate enantiomers of the reaction products.
42
Protein [Protein]
(mM)
PIX : Protein
Stoichiometry
[SYPRO
Orange]
Heating Rate Tm
(o C)
Ir(Me)-PIX
CYP119
0.1 mM 1 : 1 20 X 1o C/min 69o C
Ir(Me)-PIX
P450 BM3
0.1 mM 1 : 1 20 X 1o C/min 45o C
Ir(Me)-PIX
P450 CAM
0.1 mM 1 : 1 20 X 1o C/min 40o C
Table S7.
Melting temperatures of various Ir(Me)-PIX heme proteins, as determined by DSF using
SYPRO Orange as a fluorescent reporter.(31)
43
NMR Spectra:
44
45
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